Cells and cell culture
CRL-2097, BJ and MRC-5 and NIH/3T3 were obtained from ATCC (Manassas, VA, USA) and maintained in DMEM supplemented with 10% FBS. HBET1 [11] and their derived cells were maintained in LHC-9 medium (Thermo Fisher Scientific, Waltham, MA USA) supplemented with 2 mM L-glutamine. H3122 was obtained from the NCI Repository of Tumor Cell Lines (Frederick, MD, USA) and maintained in RPMI 1640 medium supplemented with 10% FBS. For tetracycline (Tet)-inducible gene expression, doxycycline (Dox, at 1 μg/ml) was added. In continuous culture of human fibroblasts to examine cellular replicative lifespan, the cells were passaged at a split ratio of 1:4 (or 1:2 at later passages when approaching senescence). The number of population doubling levels (PDL) achieved between passages was determined by log2 (“number of cells obtained” divided by “number of cells inoculated”) [11] and data were presented as means ± s.d. from biological triplicates. Crizotinib was purchased from Selleck Chemicals (Houston, TX, USA) and used at a concentration of 25 nM. For all cells, culture medium was changed every 48 hours. For cells treated with Dox or Dox plus Crizotinib, they were continuously included in the medium.
SA-β-gal staining was performed using the kit purchased from Cell Signaling Technology (Danvers, MA, USA).
Lentiviral and retroviral expression vectors and vector transduction
pLenti3.3/TR and a lentiviral expression vector pLenti6.3/TO/V5-DEST were from Thermo Fisher Scientific. The EML4-ALK cDNA variant 1 [3, 4] was transferred into pLenti6.3/TO/V5-DEST for its inducible (with pLenti3.3/TR) and constitutive expression (without pLenti3.3/TR). The red fluorescent protein (RFP) cDNA was also transferred from pLOC (Open Biosystem, Lafayette, CO, USA) to pLenti6.3/TO/V5-DEST, generating the control vector. EML4-ALK (K589M) in pLenti6.3/TO/V5-DEST was generated via site-directed mutagenesis using the QuikChange II XL kit (Stratagene, Carlsbad, CA, USA). The retroviral expression vector for H-RasV12 (in pBabe vector) was a gift from Dr. Manuel Serrano (IRB Barcelona, Spain) [12]. The retroviral vector for hTERT (pCLXSN-hTERT) was previously described [11] and used to generate hTERT-immortalized BJ (hTERT-BJ) and HBET1 previously [11, 13] and hTERT-transduced CRL-2097 (hTERT-CRL-2097) in this study. The preparation of vector supernatants and the vector transduction were performed as previously described [11, 14, 15]. Two days after transduction, the cells were selected with puromycin (for pBabe, 1 mg/ml; Sigma-Aldrich), G418 (for pLenti3.3/TR and pCLXSN-hTERT, 500 mg/ml; Sigma-Aldrich) or blasticidin (for pLenti6.3/TO/V5-DEST, 2 mg/ml; Thermo Fisher Scientific).
The coding sequences in all newly constructed vectors were fully sequenced for confirmation.
Protein lysates and western blot analysis
Cells were lysed in 20 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.1% SDS / 1% NP-40 / 1 mM EDTA containing the Complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Western blot analysis was performed as described previously [11, 16]. Signal detection was performed using the SuperSignal West Dura chemiluminescence substrate (Thermo Fisher Scientific) and the images were captured using the ChemiDoc Imager (Bio-Rad, Hercules, CA, USA). Quantitative image analysis used the ImageJ software (http://rsb.info.nih.gov/ij/). Primary antibodies used were as follows: anti-ALK (#3633, Cell Signaling Technology; 1:2000); anti-phosho-ALK (#3341, Cell Signaling Technology; 1:1000); anti-STAT3 (#9139, Cell Signaling Technology; 1:1000); anti-phospho-STAT3 (#9145, Cell Signaling Technology; 1:2000); anti-AKT (#9272, Cell Signaling Technology; 1:1000); anti-phospho-AKT (#9271, Cell Signaling Technology; 1:1000); anti-Src (#2123, Cell Signaling Technology; 1:1000); anti-phospho-Src (#6943, Cell Signaling Technology; 1:1000); anti-Erk1/2 (#9102, Cell Signaling Technology; 1:1000); anti-phosho-Erk1/2 (#9101, Cell Signaling Technology; 1:1000); anti-c-H-Ras (#OP23, Calbiochem; 1:30); anti-p16INK4A (sc-468, Santa Cruz Biotechnology, Dallas, TX, USA; 1:1000); anti-p21WAF1 (#2947, Cell Signaling Technology; 1:1000); anti-p53 (DO-1, sc-126, Santa Cruz Biotechnology, Dallas, TX, USA; 1:1000); anti-phospho-p53 (Ser15) (#9284, Cell Signaling Technology; 1:1000); and anti-GAPDH (sc-166574, Santa Cruz Biotechnology; 1:1000.
Immunofluorescence staining
Cells (3.0×104 cells/well) were washed with PBS and fixed for 10 min with 4% paraformaldehyde, followed by permeabilization with 0.25% Triton-X-100 for 10 min. Incubation with anti-g-H2AX antibody (Catalog #05-636, Sigma Aldrich; 1:1000) and then with an Alexa Fluor 488-conjugated secondary antibody (Catalog #A-21202; Thermo Fisher Scientific; 1:400), washing procedures, and mounting with Antifade with DAPI (Vectashield, Burlingame, California) were as previously described [17, 18]. Digital images were acquired and analyzed using confocal microscopy (Zeiss 780) and ZEN software. The total signal intensity of γ-H2AX immunofluorescence per microscopic field was determined in unmerged γ-H2AX images using Image J software and expressed as the mean signal intensity per DAPI+ nucleus manually quantified in five high powered fields (40x).
Anchorage-independent colony formation in soft-agar medium
Cells (1,000 cells/well) were seeded on 12-well plates in 1 ml of medium containing 0.35% agarose (SeaPlaque low-melting-temperature agarose, Lonza Biosciences, Alpharetta, GA, USA) over 1-ml volume of base layer consisting of the same culture medium and 0.5% agarose. Colonies of approximately 50 mm in diameter or larger were counted on day 21.
Telomere length measurement by quantitative PCR (qPCR)
Telomere length was measured by the monochrome multiplex qPCR (MMQPCR) method as described previously [19]. Relative telomere length was express as a ratio of the quantity of telomeric DNA (T) normalized to the quantity of multiple copy sequence DNA (mcs), yielding T/M ratio. Intra- and inter-assay coefficient of variations (CVs) were 4.1% and 10.7%, respectively.
G-band karyotyping and spectral karyotyping (SKY)
Treatment with Colcemid (10 mg/ml; KaryoMax, Invitrogen, Carlsbad, CA, USA), hypotonic treatment (0.075 M KCl), fixation with methanol/acetic acid (3:1), slide preparation and G-banding were performed as previously described [20] and the images were captured and analyzed with the HiBand system (Applied Spectral Imaging, Carlsbad, CA, USA). For spectral karyotyping (SKY), the slides were processed using the 24-color Human SKY Paint kit (Applied Spectral Imaging) according to the manufacturer’s protocol. Spectral images of the hybridized metaphases were acquired using the HyperSpectral Imaging system (Applied Spectral Imaging) and analyzed using the HiSKY v.7.2 acquisition software (Applied Spectral Imaging). All cell lines were analyzed by G-band karyotyping. hTERT-CRL-2097, hTERT-CRL-2097+EML4-ALK, its derived soft-agar clones #1 and #2, and HBET1 were also analyzed by SKY.
Tumorigenicity in vivo
Female NOD.SCID/Ncr mice were obtained from Charles River Laboratories (#560; Germantown, MD, USA), and were maintained under specific pathogen-free conditions and the animals had free access to feedstuff and water under 12:12 light/dark cycles, 22 and 30 – 70 % humidity. These mice at 6 to 10 weeks of age were subcutaneously injected with cells (5 ´ 106 per flank) in 50% Matrigel (no. 354248, Corning, NY, USA) at both flanks: one side with EML4-ALK-expressing cells or their derived soft-agar clones; and the other side with vector control cells. For injection of these cells, mice were anesthetized using isoflurane in accordance with NCI Animal Care and Use Committee (ACUC) guidelines. As a positive control, NIH/3T3 cells expressing EML4-ALK were injected at both flanks of two mice (2 ´ 106 per flank). Tumor size and body weight were measured twice a week until 8 weeks or tumor size excess 20 mm. To ameliorate the suffering of mice observed throughout experimental studies, mice were euthanized by CO2 inhalation. After 8 weeks of administration, the mice were sacrificed and photographed, and the tumors were removed and weighed. This experiment was approved by the NCI ACUC (LHC-012-D).
RNA isolation, reverse transcription (RT)-PCR and Sanger sequencing of the entire coding region of p53 mRNA
Total RNA isolation, reverse transcription, and 1st strand cDNA synthesis with random hexamers were carried out as previously described [11, 15]. PCR amplification of the entire coding region of p53 mRNA was carried out using the Platinum Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific) with the primers 5’-ATG GAG GAG CCG CAG TCA-3’ and 5’-GTC AGT CTG AGT CAG GCC CTT C-3’. The amplified PCR product was sequenced using the BigDye Terminator v1.1 Cycle Sequencing kit (Thermo Fisher Scientific) with 5’-AGT ACG TGC AAG TCA CAG-3’, 5’-CGT CCC AAG CAA TGG ATG-3’ and 5’-CTC ACC ATC ATC ACA CTG G-3’.
RNA sequencing (RNA-seq) analysis
RNA samples were treated with DNase I (no. 18068015, Thermo Fisher Scientific), followed by purification with RNeasy MinElute Cleanup kit (Qiagen). The RNA integrity numbers (RIN) of the samples on the Agilent 2100 Bioanalyzer (Agilent Technologies) were 9.3 to 10.0. All the following steps of the RNA-seq experiment were carried out at the CCR Sequencing Facility (Leidos Biomedical Research, Frederick, MD, USA). Sequencing libraries were prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina, San Diego, CA, USA) and were sequenced with paired-end reads of 150 bp on HiSeq3000/4000 sequencer (Illumina) to obtain at least 30 million read pairs per sample. Reads were trimmed for adaptors and low-quality bases using Trimmomatic [21] and were aligned with the reference genome (Human-hg38) and the annotated transcripts (Gencode_v24) using STAR (https://github.com/alexdobin/STAR). The gene expression quantification analysis was performed using STAR/RSEM tools (http://deweylab.github.io/RSEM/) to obtain raw read counts and normalized read counts (RPKM) for each gene.
Differentially expressed genes were identified using DESeq2 in R [22] with a false discovery rate (FDR) cutoff of 0.01, and analyzed by gene enrichment analysis for KEGG (https://www.genome.jp/kegg/) and Reactome Pathways (https://reactome.org/) using DAVID v6.8 (https://david.ncifcrf.gov/). Enriched pathways were identified according to FDR £ 0.05.
MicroRNA quantification by qPCR
PCR was performed by TaqMan MicroRNA Assays as manufacturer’s instructions. Primers used were as follows: miR-21 (Taqman microRNA assay, hsa-miR-21, PN 4440887); RNU48 (Taqman microRNA assay, RNU48, PN 4440887).
Data mining of publicly available lung adenocarcinoma datasets
For the TCGA dataset [23], mRNA expression from RNA Seq V2 RSEM and tumor characteristics were downloaded from cBioPortal for 510 patients with lung adenocarcinoma. Log2-transformed RSEM values for hTERT were used for the analyses.
For the microarray dataset GSE31210 [24], data and sample characteristics were downloaded from the Gene Expression Omnibus for 226 patients with lung adenocarcinoma using the R package GEOquery. The raw intensity values were processed and normalized with Robust Multi-Array Average (RMA) using the R package oligo. Affymetrix IDs were mapped to HUGO Gene Nomenclature Committee IDs. Resulting RMA expression values for hTERT were used for the analyses.
Quantitative RT-PCR analysis
Quantitative RT-PCR analysis to confirm the RNA-seq data was performed by using TaqMan Assay as manufacture’s instruction. Primer used was as follows: IL1B (Taqman Hs01555410_m1, Cat 4331182), CXCL8 (Taqman Hs00174103_m1, Cat 4331182), GAPDH (Taqman Hs02786624_g1, Cat 4331182), PLAU (Taqman Hs01547050_m1, Cat 4331182), PLAT (Taqman Hs 00938315_m1, Cat 4331182) and A2M (Taqman Hs 00929971_m1, Cat 4331182).
Statistical analysis
Data are presented as means ± s.d. from at least three biological replicates. Statistical significance was evaluated using unpaired 2-tailed Student’s t-test. * P < 0.05, ** P < 0.01 and *** P < 0.001.